The exocrine secretion of the acinar gland cells in the human prostate consists of, among other components, a serous secretion and prostasomes. The prostasomes are functionally associated with both reproduction and prostate cancer development and are capable to raise autoantibodies at various pathologies. Therefore, we are trying to characterize prostasome antigens by analysing prostasome- producing cell lines of prostate cancers with the cDNA microarray technique. To obtain one state with synthesis of prostasomes and another state without synthesis, we checked whether the prostasome differentiation was influenced by the mode of growing the cells, that is, whether the cells had been growing on a solid support or on a flexible one.We studied the expression of prostasomes in the cell lines PC3, DU145 and LNCaP. We grew the cells with the following methods: Monocellular layers on microbeads, multicellular spheroids, single cells in suspension cultures, and xenotransplants in nude rats. The presence of prostasomes was examined by ELISA, immunocytochemistry or electron microscopy.The results showed that growing the cells on microbeads (solid support) produced a differentiation of prostasomes, while growing the cells in multicellular spheroids (flexible support) did not. Thus it should be possible to apply cDNA microarray analyses for characterizing the genes which are active at the cellular expression of prostasomes and then deduce the prostasome antigens.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

Prostate cancer consistently remains a difficult clinical enigma. Therefore, the development of novel strategies for diagnosis and treatment (e.g. immunotherapy) of prostate cancer is essential. We tried to identify the prostasome-derived proteins that were immunogenic in prostate cancer patients. Prostate cancer patients’ sera (n 5 44) with high enzyme-linked immunosorbent assay (ELISA) titers against prostasomes were selected for immunoblotting against purified seminal prostasomes. The SDS-PAGE and immunoblotting experiments were performed with Bio-Rad systems. Twenty-five of the recognized proteins were isolated and analyzed by means of mass spectrometry. Out of 44 patients’ sera, 31 (70%) demonstrated in immunoblotting experiments reactivity against several prostasomal protein bands in the molecular weight range of 10– 200 kDa. Some of the bands (55, 70 and 170 kDa) were more frequently recognized by the patients’ sera. Concomitantly run control sera generated only very weak or no bands at all. The most frequently occurring prostasomal proteins were identified as heat shock proteins (HSP 70, 71) and clusterin. This study identified the most important molecular targets of autoantibodies against prostasomes generated in connection with the development of prostate cancer in man. These immunogenic prostasomal proteins could be appropriate target molecules for specific immunotherapy of prostate cancer patients.

Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins.

BACKGROUND:Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called "storage vesicle" equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization.METHODS:Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species.RESULTS:The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10-150kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes.CONCLUSION:These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum.GENERAL SIGNIFICANCE:This study unravels energy metabolic relationships of prostasomes from four different species.